Molecular genetic identification of Japanese sand lances using mitochondrial DNA cytochrome c oxidase subunit 1 restriction fragment length polymorphisms

We developed a novel molecular genetic method using restriction fragment length polymorphisms (RFLPs) to identify the taxonomically debatable Japanese sand lances of the genus Ammodytes. Partial nucleotide sequences of the mitochondrial DNA cytochrome c oxidase subunit 1 (COI) gene of Ammodytes japonicus (n = 405), Ammodytes heian (n = 284), and Ammodytes hexapterus (n = 15) were determined and found to contain species-specific restriction sites for HindIII-HF and RsaI enzymes. The observed RFLP patterns from a combination of two enzymes were specific in each species except for specimens with rare haplotypes of A. japonicus (1.5 %) and A. heian (2.1 %), whereas detailed morphological analysis using 29 characters failed to discriminate the COI-identified specimens of these two species collected from the same locality. The developed molecular method will allow rapid and precise identification of the Japanese sand lances, especially for the sympatric A. japonicus and A. heian, thereby providing a reliable means for ecological research and stock management of these species.